The long term goal of this proposal is to completely reconstitute the HIV replicative reaction in vitro, starting with model viral RNA and finishing with an authentic viral DNA nucleoprotein particle that is capable of integrating efficiently into exogenous DNA. In reconstituting these reactions we will employ the p5l and p66 subunits of HIV reverse transcriptase, HIV nucleocapsid protein, HIV integration endonuclease, natural and synthetic priming tRNA(lys)3 and model HIV templates generated in vitro using T7 RNA polymerase. The reactions will also contain any additional viral or host factors for which a requirement is found. Such a requirement will form the basis of a functional assay to guide the purification of these factors. Mechanistic studies will focus on (i) the asymmetric contributions of the p5l and p66 subunits to the retroviral reaction, (ii) the role of the 1Me-A residues 19 nucleotides in from the 3'-terminus of tRNA(lys)3 in determining the (+) strand strong stop intermediate and (iii) the role of nucleocapsid protein in strand transfer reactions. The mechanistic insight provided by these studies may provide additional avenues to the rational design of anti-HIV therapeutic agents.